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pEarleyGate 301

Gateway®-compatible promoterless plant transformation vector with a C-terminal HA tag.

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pEarleyGate 301 Sequence and MappEarleyGate 301.dna
Map and Sequence File   
Sequence Author:  Pikaard Lab
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 AseI (94) BclI * (10,142) PsiI (10,011) BspHI (9685) EcoRV (9262) NsiI (9151) PluTI (8003) SfoI (8001) NarI (8000) KasI (7999) EcoNI (7527) BseRI (7491) BmtI (7218) NheI (7214) BsiWI (7098) XmnI (95) Eco53kI (103) SacI (105) FspAI (523) AarI (587) AfeI (760) BspEI (1806) NcoI (2111) MluI (2257) BbvCI (2535) SrfI (2681) BstXI (2798) HA XbaI (3109) PacI (3122) SpeI (3134) MfeI (3438) BstEII (3710) AhdI (3840) SbfI (3865) HindIII (3873) PvuI (3999) PmeI (4086) pEarleyGate 301 10,379 bp
AseI  (94)
1 site
A T T A A T T A A T T A
BclI  (10,142)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
PsiI  (10,011)
1 site
T T A T A A A A T A T T
BspHI  (9685)
1 site
T C A T G A A G T A C T
EcoRV  (9262)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
NsiI  (9151)
1 site
A T G C A T T A C G T A
PluTI  (8003)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (8001)
1 site
G G C G C C C C G C G G
NarI  (8000)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (7999)
1 site
G G C G C C C C G C G G
EcoNI  (7527)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BseRI  (7491)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
BmtI  (7218)
1 site
G C T A G C C G A T C G
NheI  (7214)
1 site
G C T A G C C G A T C G
BsiWI  (7098)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
XmnI  (95)
1 site
G A A N N N N T T C C T T N N N N A A G
Eco53kI  (103)
1 site
G A G C T C C T C G A G
SacI  (105)
1 site
G A G C T C C T C G A G
FspAI  (523)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (587)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
AfeI  (760)
1 site
A G C G C T T C G C G A
BspEI  (1806)
1 site
T C C G G A A G G C C T
NcoI  (2111)
1 site
C C A T G G G G T A C C
MluI  (2257)
1 site
A C G C G T T G C G C A
BbvCI  (2535)
1 site
C C T C A G C G G A G T C G
SrfI  (2681)
1 site
G C C C G G G C C G G G C C C G
BstXI  (2798)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
XbaI  (3109)
1 site
T C T A G A A G A T C T
PacI  (3122)
1 site
T T A A T T A A A A T T A A T T
SpeI  (3134)
1 site
A C T A G T T G A T C A
MfeI  (3438)
1 site
C A A T T G G T T A A C
BstEII  (3710)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
AhdI  (3840)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
SbfI  (3865)
1 site
C C T G C A G G G G A C G T C C
HindIII  (3873)
1 site
A A G C T T T T C G A A
PvuI  (3999)
1 site
C G A T C G G C T A G C
PmeI  (4086)
1 site
G T T T A A A C C A A A T T T G
pVS1 RepA
6483 .. 7556  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas
plasmid pVS1
pVS1 RepA
6483 .. 7556  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas
plasmid pVS1
KanR
9161 .. 9955  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
9161 .. 9955  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
OCS terminator
3150 .. 3857  =  708 bp
octopine synthase terminator
OCS terminator
3150 .. 3857  =  708 bp
octopine synthase terminator
CmR
1597 .. 2256  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1597 .. 2256  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
pVS1 StaA
5425 .. 6054  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas
plasmid pVS1
pVS1 StaA
5425 .. 6054  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas
plasmid pVS1
ori
8486 .. 9074  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8486 .. 9074  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
BlpR
375 .. 926  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
BlpR
375 .. 926  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
MAS promoter
932 .. 1312  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
MAS promoter
932 .. 1312  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
ccdB
2598 .. 2903  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
2598 .. 2903  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
MAS terminator
113 .. 365  =  253 bp
mannopine synthase terminator
MAS terminator
113 .. 365  =  253 bp
mannopine synthase terminator
pVS1 oriV
7622 .. 7816  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
pVS1 oriV
7622 .. 7816  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
bom
8160 .. 8300  =  141 bp
basis of mobility region from pBR322
bom
8160 .. 8300  =  141 bp
basis of mobility region from pBR322
attR2
2944 .. 3068  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2944 .. 3068  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1364 .. 1483  =  120 bp
recombination site for the Gateway® LR reaction
attR1
1364 .. 1483  =  120 bp
recombination site for the Gateway® LR reaction
lac UV5 promoter
1513 .. 1543  =  31 bp
   Segment 1:  -35  
   1513 .. 1518  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1513 .. 1543  =  31 bp
   Segment 2:  
   1519 .. 1536  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1513 .. 1543  =  31 bp
   Segment 3:  -10  
   1537 .. 1543  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1513 .. 1543  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
HA
3070 .. 3096  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
3070 .. 3096  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
LB T-DNA repeat
3 .. 27  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
3 .. 27  =  25 bp
left border repeat from nopaline C58 T-DNA
RB T-DNA repeat
4101 .. 4125  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
4101 .. 4125  =  25 bp
right border repeat from nopaline C58 T-DNA
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